Identifying where HIV persists in HIV-infected patients on suppressive therapy is a critically important step towards HIV eradication. For practical reasons, the study of viral reservoirs has largely focused on components of peripheral blood. Recent findings, however, show that tissue sites harbor a substantial proportion of infected cells. The overall goal of this Project is to identify the source, dynamics, and nature of the reservoir producing persistent HIV infection in patients on suppressive therapy in a range of tissue sites. The research team will determine the nature of HIV persistence/latency in T-cell subsets (naive, memory, central memory and effector/transitional memory) and hematopoietic progenitor cells from the small bowel, large bowel, lymph nodes, and bone marrow of patients on long-term therapy (>7 years) who initiated therapy during acute and chronic infection. We will also investigate HIV in rare circulating cells (which will be obtained from leukapheresis). Unique and innovative techniques will be used to (1) analyze the genetic make-up of HIV populations in the cell subsets, (2) quantify the levels of intracellular HIV DNA and unspliced RNA/spliced RNA and, using a novel nucleic acid chemistry for primer-probe design, measure short abortive HIV transcripts, (3) determine the replication competence of the HIV remaining in different cellular subsets, (4) reveal host cell factors that determine which cells may harbor or resist replicating and/or latent HIV and (5) examine the effect of collagen deposition and fibrosis on the size and distribution of the reservoirs. We will also support complementary work being done in tissue-based macrophages (Project 4). In addition to understanding how HIV is subdivided among different cells and tissues, the proposed study will provide a systematic survey of how lymphoid cell host factors and changes in lymph node tissue structure support HIV latency and help determine the magnitude and nature of the viral reservoirs. We believe that this study will provide an unprecedented quantitative assessment of total body stores of virus and that our findings will guide treatment interventions that can reduce and eradicate persistent HIV reservoirs.